Single-cell transcriptomics unveils xylem cell development and evolution

Background Xylem, the most abundant tissue on Earth, is responsible for lateral growth in plants. Typical xylem has a radial system composed of ray parenchyma cells and an axial system of fusiform cells. In most angiosperms, fusiform cells comprise vessel elements for water transportation and libriform fibers for mechanical support, while both functions are performed by tracheids in other vascular plants such as gymnosperms. Little is known about the developmental programs and evolutionary relationships of these xylem cell types. Results Through both single-cell and laser capture microdissection transcriptomic profiling, we determine the developmental lineages of ray and fusiform cells in stem-differentiating xylem across four divergent woody angiosperms. Based on cross-species analyses of single-cell clusters and overlapping trajectories, we reveal highly conserved ray, yet variable fusiform, lineages across angiosperms. Core eudicots Populus trichocarpa and Eucalyptus grandis share nearly identical fusiform lineages, whereas the more basal angiosperm Liriodendron chinense has a fusiform lineage distinct from that in core eudicots. The tracheids in the basal eudicot Trochodendron aralioides, an evolutionarily reversed trait, exhibit strong transcriptomic similarity to vessel elements rather than libriform fibers. Conclusions This evo-devo framework provides a comprehensive understanding of the formation of xylem cell lineages across multiple plant species spanning over a hundred million years of evolutionary history. Supplementary Information The online version contains supplementary material available at 10.1186/s13059-022-02845-1.

Fig. S1. Summary of scRNA-seq assays of SDX in P. trichocarpa, E. grandis, T. aralioides and L. chinense. Statistics of scRNA-seq profiling by 10x Chromium (red) or MARS-seq2.0 (green) are shown for cells with at least 500 and 100 UMIs, respectively, including cell numbers, biological replicate numbers, total detected genes, percentage of detected genes, mean read counts per cell and median number of genes detected per cell.   Scale bar, 500 μm. In (C) and (D), the area switched from red to white, leaving an empty space after the libriform fibers were cut by laser. Libriform fibers in transverse and tangential views (E-I). A transverse section with an area highlighted (red) for libriform fiber collection (E). A closeup of the highlighted area (F). Three-dimensional structure around the highlighted area (G). A tangential section with highlighted area (H). Corresponding panel of Fig. 1E (I).

Tangential scheme
Transverse Tangential   Transverse  scheme   Tangential  scheme   Transverse   Tangential Transverse scheme S6. The illustrations for vessel element and ray parenchyma cell collection from transverse and tangential perspectives. (A-C) The schematics of vessel elements from transverse and tangential perspectives. The blue area represents the vessel elements collected using LCM. In (A), the corresponding figure of Fig. 1I represents a tangential section with an area highlighted (blue) for vessel element collection. In (B), a threedimensional structure of the highlighted area shows the location of the collected vessel elements in the stem structure schematic. In (C), the corresponding figure of Fig. 1H represents a transverse section with highlighted area. (D-F) The schematics of ray parenchyma cells from transverse and tangential perspectives. The pink area represents the ray parenchyma cells collected using LCM. In (D), the corresponding figure of Fig.  1M represents a tangential section with a highlighted area (pink). In (E), a threedimensional structure of the highlighted area shows the location for the collected ray parenchyma cells in the stem structure schematic. In (F), the corresponding figure of Fig.  1L represents a transverse section of the highlighted area. (G and H) The threedimensional arrangement of three xylem cell types from tangential (G) and radial (H) perspectives. F, libriform fibers. V, vessel elements. R, ray parenchyma cells. Potri.003G188500

Ray cell lineage
Transcript abundance low high

Ptr8
Transcript abundance low high

Fig. S22. Transcript abundance of marker genes of each cell cluster in P. trichocarpa.
Many DEGs from each cell cluster are identified as marker genes if these genes show an exclusive expression in that cell cluster.  Principal component analysis and differential expression analyses were conducted using the SDX RNA-seq datasets from different internodes. Total 23,441 genes were expressed in secondary xylem, and 12/6/11/11/18 genes were differentially expressed in sections 1 to 5, respectively. XE, secondary xylem expressed genes. S1 to S5, sections 1 to 5.

P. trichocarpa E. grandis
Transcript abundance low high  P. trichocarpa and E. grandis. (A) The cell cluster plots obtained through unsupervised K-means clustering and UMAP are based on the SDX scRNA-seq results of P. trichocarpa and E. grandis, respectively. (B) The clusterexclusive distributions of each marker orthologous group are represented by Group #1287 for vessel element/late fusiform precursor (blue), Group #316 for ray organizer (orange), Group #1373 for fusiform early precursor (green), Group #132 for ray precursor (yellow), Group #1385 for fusiform organizer (purple), Group #9398 for libriform fiber (red) and Group #180 for ray parenchyma cell (pink). No marker orthologous groups are observed in fusiform intermediate precursor (brown).   . trichocarpa and T. aralioides. (A) The cell cluster plots obtained through unsupervised K-means clustering and UMAP are based on the SDX scRNA-seq results of P. trichocarpa and T. aralioides, respectively. (B) The clusterexclusive distributions of each marker orthologous group are represented by Group #1848 for vessel element/late fusiform precursor (blue), Group #9334 for fusiform early precursor (green), Group #4229 for fusiform organizer (purple) and Group #1916 for ray parenchyma cell (pink). No marker orthologous groups are observed in fusiform intermediate precursor (brown), ray organizer (orange) and ray precursor (yellow).   . Single-species unsupervised K-means clustering (i-iii). Two-species graph-based cell clustering using orthologous genes (iv-vii). In (i) and (v), black dots are SDX cells from P. trichocarpa. In (iii), (iv), (v), and (vii), gold dots are cells from A. thaliana or O. sativa. In (iv), grey dots represent the SDX cells from P. trichocarpa, and the xylem cells identified in previous Arabidopsis or rice studies are in magenta. In (ii) and (vii), the colors of cell clusters are based on the single-species cell clustering results. The cell clusters in twospecies clustering (vi).

UMAP2
Transcript abundance low high Group #2901

T5L1/BHLH30, TMO5
Group #6149      Fig. S36. Two-species clustering and visualization of scRNA-seq data between P. trichocarpa and P. alba var. pyramidalis. Two-species clustering of SDX single cells in P. trichocarpa and P. alba var. pyramidalis. Single-species unsupervised K-means clustering (i-iv). Two-species graph-based cell clustering using orthologous genes (v-vii). In (i), (iii), and (v), black dots are SDX cells from P. trichocarpa and gold dots are cells from P. alba var. pyramidalis. In (ii), the colors of cell clusters in P. trichocarpa are based on the single-species cell clustering results. In (iv), the cell cluster colors are assigned using the co-located colors from P. trichocarpa cell clusters. The cell clusters in two-species clustering (vi). In (vii), the colors of two-species clustering are derived from that of single-species clustering.